An unusual case of autoimmune agranulocytosis with total absence of myeloid precursors: demonstration of diverse sources of R binder for cobalamin in plasma and secretions

An unusual autoimmune agranulocytosis featured intense plasmacytosis, hyperglobulinemia, and total absence of myeloid precursors. Remission was induced with prednisone and cyclophosphamide on two separate occasions, producing reversal of all the above abnormalities. Anti-granulocyte antibodies were demonstrated in relapse by staphylococcal protein A assay. During agranulocytosis, levels of the R binder for cobalamin in the blood were greatly diminished (20-60 pg/mL unsaturated binding capacity). Moreover, the acquired R binder deficiency was accompanied by the previously undescribed phenomenon of a shift of endogenous serum cobalamin to transcobalamin II; only 10-19% of endogenous cobalamin was attached to R binder. In remission, unsaturated R binder rose to normal levels and R binder also carried 78-91% of endogenous cobalamin. This confirms that granulocytes are responsible for most of the circulating R binder in humans. In contrast, salivary R binder concentration was normal throughout the patient's course, indicating that secretory R binder is nongranulocytic in origin. These data establish that R binder has more than one source in humans.     

Comparison of human chorionic gonadotropin +beta and invasive trophoblast antigen disappearance rates in serum after evacuation of molar pregnancy

After the evacuation of a hydatidiform mole, the spontaneous regression or the persistent trophoblastic disease (PTD) needing chemotherapy, is monitored by determining the serum human chorionic gonadotropin (hCG) concentration. Hyperglycosylated hCG (invasive trophoblast antigen, ITA) has been suggested to be of clinical value in the diagnosis and follow-up of gestational trophoblastic disease including PTD. To further document the relationship between ITA and hCG in spontaneous post-molar regression and during chemotherapy treatment of PTD, we used distinct immunoassays to measure the concentrations of hCG+beta and ITA in serum from three groups of patients after the evacuation of moles. For each group [uneventful post molar hCG regression, group 1; PTD treated with Methotrexate (MTX) (mono-chemotherapy), group 2; and PTD with MTX and poly-chemotherapy (EMA-CO), group 3], we compared the time course of the serum concentrations after evacuation, and determined the disappearance rates (half-lives) within and between treatment groups. Significantly longer mean serum half-lives for hCG+beta and ITA were found in the poly-chemotherapy (group 3: 3.02 and 2.51 weeks) as compared to the mono-chemotherapy group (group 2: 0.96 and 0.90 weeks) and the uneventful regression group (0.81 and 0.66 weeks) (each, p=0.003), but no differences were observed between the mono-chemotherapy and the uneventful regression group. Significantly shorter mean half-lives for ITA than those calculated for hCG+beta were observed in all three groups of patients. The implication and the possible clinical value of the more rapid regression of ITA to baseline levels as compared to hCG+beta remain to be investigated prospectively.     

Modulation of placental vascular endothelial growth factor by leptin and hCG

Vascular endothelial growth factor (VEGF) has been identified as an endothelium-specific mitogen and inducer of angiogenesis and endothelial cell survival. Leptin and hCG have also been suggested as possible regulators of angiogenesis in various models. In-vivo and in-vitro assays revealed that leptin has an angiogenic activity and that the vascular endothelium is a target for leptin. Thus, we hypothesized that products of cytotrophoblastic cells may play a role in placental angiogenesis and we therefore investigated the effects of leptin and hCG on cytotrophoblast VEGF secretion. We incubated cytotrophoblastic cells (CTB) with recombinant human leptin (rhLept) (0-4 pg/ml) or hCG (0-30000 IU/ml) for 4 h. rhLept significantly stimulated hCG (P = 0.0045) and decreased VEGF release (P = 0.0008) by CTB in a concentration-dependent manner. On the other hand, increasing concentrations of hCG (0-30000 IU/ml), induced a significant inhibition of leptin secretion (P = 0.0028) and a marked dose-dependent stimulation of VEGF(165) secretion (P < 0.0001). We observed an increase of >1000-fold in basal trophoblastic VEGF secretion with physiological concentrations of hCG in vitro. An inhibitory effect of hCG on trophoblastic leptin secretion was also observed, suggesting that hCG might exert a possible negative feedback on trophoblastic release of leptin. We hypothesize that trophoblastic products such as hCG and leptin are probably involved in the control of VEGF secretion at the maternal-fetal interface.     

The Effect of Disodium Cromoglycate on In Vitro Mast Cell Degranulation in Human Jejunal Mucosa

The in vitro effect of disodium cromoglycate (DSCG) on IgE antigen-induced mast cell degranulation is described. Serum from an egg white allergic patient was used to sensitize jejunal mucosa from nine individuals. Egg white was used to challenge the IgE sensitized mast cells. DSCG in concentrations 3 x 10(-7) M to 3 x 10(-4) M was added to the mucosal specimens before antigen challenge. Mast cell degranulation in the sensitized specimens challenged with egg white was 38%. Mast cell degranulation in sensitized specimens treated with DSCG before and during antigen challenge was reduced to 2% at a concentration of 3 x 10(-5) M of DSCG (P=0.006) and to 28% at a concentration of 3 x 10(-6) M (P=0.027). No significant reduction of mast cell degranulation was seen at concentrations of 3 x 10(-7) M and 3 x 10(-4) M. The results support a role for DSCG in the treatment of gastrointestinal allergy.     

Endometrial adenocarcinoma associated with elevated serum concentrations of the free beta subunit of human chorionic gonadotropin

We report a case of a histologic grade II endometrial adenocarcinoma without trophoblastic differentiation in a 24-year-old woman with an elevated serum concentration of human chorionic gonadotropin (hCG) and with no evidence of pregnancy. Serum and urine specimens were used to study the hCG immunoreactivity. Qualitative tests performed on serum and urine using 5 different assays produced conflicting results. The hCG concentration in serum and urine was quantified using assays designed to detect different molecular forms of the molecule; analysis revealed that serum hCG immunoreactivity was due entirely to the presence of the free beta subunit. Immunohistochemical analysis performed on tissue samples showed strong cytoplasmic staining for hCG. While hCG is a well-recognized tumor marker in gynecologic malignant neoplasms, immunoreactivity most often is due to the presence of both intact molecule and the free beta subunit. To our knowledge, this is the first report of an endometrial adenocarcinoma producing only the free beta subunit of hCG.     

Monitoring human chorionic gonadotrophin level: evaluation of urine as an alternative specimen type

Although urine has been used widely for the qualitative detection of human chorionic gonadotrophin (hCG), serum is chosen conventionally for the serial quantification of the hormone to monitor trophoblastic activity. In response to requests from both clinicians and patients regarding the use of urine as an alternative specimen type, we designed this comparative study to evaluate the possibility, taking into account both laboratory technique and the distribution of hCG within different body fluids. Using the Access Chemiluminescent Immunoassay System, total beta-hCG was measured in serum and urine (n = 30) collected from patients hospitalised for first-trimester abnormal pregnancy. Results obtained with normalised urine (corrected with urinary creatinine) and serum total beta-hCG correlated well (r = 0.98, P < 0.001), and we concluded that urine could be used as an alternative specimen type for the serial quantitation of hCG to monitor trophoblastic activity. However, the assay used must detect the common beta 2 epitope.     

Monoclonal antibody specific to the beta subunit of human chorionic gonadotropin (hCG) and capable of agglutinating hCG-coated red blood cells

Spleen cells of BALB/c mice immunized with human chorionic gonadotropin (hCG) were fused with NS-1 mouse myeloma cells. A hybrid cell line, clone PE4 secreting monoclonal antibody (MAb) to hCG was isolated by simultaneous screening with both a radioimmunoassay (RIA) and a hemagglutination inhibition assay (HAI). With the aid of the double-antibody radioimmunoassay, it was established that PE4-MAb recognizes the beta-subunit of hCG. It shows an affinity constant of 0.4 X 10(10) L/M and cross-reactivity of 0.1% to other related human glycopeptide hormones. PE4-MAb agglutinates sheep red blood cells coated with hCG and can be used in an HAI assay for hCG. Dual screening procedures have, thus, led to a monoclonal antibody showing high sensitivity in two different assays, and hence useful for the qualitative detection and quantitative determination of hCG by both RIA and HAI methods.     

Characterization of autoantibodies from patients with Goodpasture's disease using a resonant mirror biosensor

Goodpasture's disease is characterized by the binding of IgG autoantibodies to the glomerular basement membrane, leading to glomerular inflammation. The autoantigen has been identified as the noncollagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). We have used the IAsys resonant mirror biosensor to analyse the extent and affinity of binding of anti-GBM antibodies from sera of patients to purified alpha3(IV) NC1. alpha3(IV) NC1 monomers were immobilized to a carboxylate cuvette, with the simultaneous use of a control well. The binding of serum from patients with Goodpasture's disease (n = 12), normal controls (n = 14) and disease controls with vasculitis (n = 14) was analysed. Antibody binding was detected in sera from all patients with Goodpasture's disease but not from controls. IAsys measurements of binding correlated with antibody levels assessed by the standardized ELISA used for clinical assays. Both ELISA and biosensor measurements showed declining antibody levels in serial serum samples from treated patients; however, the biosensor detected antibody recrudescence when ELISA remained negative. Autoantibodies from patients' serum had average affinity constants (Kd) of 6.5 x 10-11M to 52.07 x 10-10M, as determined by an inhibition assay, indicating high affinity. Sips analysis showed that the antibody response was relatively homogeneous (values of 0.46-1). Biosensor techniques can therefore be used to detect and characterize anti-GBM antibodies in serum from patients, with high sensitivity and without need for antibody purification. This technique may be useful in diagnosis and monitoring of patients with Goodpasture's disease, and may be applicable to other autoantibody mediated diseases.     

Placental site trophoblastic tumor in a postmenopausal woman.

Placental site trophoblastic tumor is a rare neoplasm that arises in the trophoblastic tissue of the placental bed. This case report is unusual because of the patient's advanced age at the time of diagnosis and the favorable response of the disease to chemotherapy. Although the clinical course is benign for most patients with placental site trophoblastic tumor, the malignant variant of the disease is characterized by recurrence, relative insensitivity to radiation and chemotherapy, and death. To the authors' knowledge, the 53-year-old woman reported is the oldest patient with histologically confirmed placental site trophoblastic tumor. Initially, surgery, radiation, and multiagent chemotherapy failed to control vaginal and pulmonary metastatic disease. After administration of four treatment cycles of a "second-line" chemotherapeutic regimen consisting of cyclophosphamide and cisplatin, complete clinical and radiologic remission was achieved. The patient's serum level of human chorionic gonadotropin has remained undetectable, and she has been without measurable evidence of disease for 16 months.     

PLAGENTAL-SITE TROPHOBLASTIC TUMOR OF THE UTERUS: Clinicopathological and Immunohlstochemical Observations of a Case

The clinicopathological features of a 28-y-ear-old woman with placental-site trophoblastic tumor (PSTT) are described. The patient presented with severe proteinuria and was found to have a cystic uterine tumor. The serum β- human chorionic gonadotropin (hCG) level was only slightly elevated. The tumor extended to the serosa without gross metastasis, and was resected. The specimen was composed of active intermediate trophoblasts (IT) and degenerative or inactive ITs. The former component had round to oval and vesicular nuclei, and abundant amphophilic or lightly eosinophilic cytoplasm. The latter component had irregular-shaped pyknotic nuclei and deeply eosinophilic cytoplasm. However, the tumor lacked the bilaminar (cyto- and syncytio-trophoblastic) structure that is a characteristic feature of choriocarcinoma. Immunohistochemical evaluation with human placental lactogen (hPL) and hCG antisera revealed that most of the tumor cells contained abundant hPL, whereas only a small number of cells contained hCG. This method seemed to be most helpful for the differential diagnosis of PSTT from other trophoblastic tumors or non-trophoblastic uterine tumors, and also to be useful for determining the prognostic behavior of PSTT.     

Short-loop feedback control of luteinizing hormone in the rabbit.

A radioimmunoassay for rabbit luteinizing hormone (rLH) in which rLH shows no significant cross-reaction with human LH (hLH) or human chorionic gonadotropin (HCG) was employed to test for the existence of a short-loop feedback for LH in the rabbit. Two weeks after castration, hCG and hLH were administered intravenously to rabbits, and the effects on circulating rLH were measured. Purified hLH (10 ng or 100 IU) produced significant depression of blood rLH within 30-60 min of intravenous injection. Saline administered to the same animals produced no changes in rLH. Injection of hCG (2,000 IU) under the same conditions also produced a significant fall in rLH. However, hCG administered to rabbits castrated 6 wk prior to study failed to suppress endogenous rLH. These data demonstrate, by direct radioimmunoassay quantification of blood hormones, the existence of a short-loop negative feedback for LH in the rabbit. They also suggest that the sensitivity of the short-loop changes with time after castration.     

Cutaneous metastasis of choriocarcinoma: a case report

Choriocarcinoma is one of the malignant tumors of trophoblastic cells characterized by the secretion of human chorionic gonadotrophin (hCG) (1-3). Cutaneous metastasis is a rare presentation of choriocarcinoma but a poor prognostic sign because it is associated only with widespread disease (3-5). A 52-yr-old female complaining of dyspnea for 2 months, presented with fingertip sized erythematous nodules on the left side of the neck and the right side of the upper back of 1 month duraton. She has suffered from Behcet's disease since 1999. Microscopic examination of a nodule of upper back demonstrated biphasic pattern of cytotrophoblasts and hCG-positive syncytiotrophoblasts, and the typical histologic features of choriocarcinoma. She was referred to the gynecological oncology department. After 17 cycles of combination chemotherapy, the serum hCG level has fallen from 700,000 to under 2.0 mIU/mL and the skin lesions have almost disappeared. However, after 3 months, total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed due to elevated serum hCG level (4,447.7 mIU/mL), and she is scheduled to receive post-operative adjuvant chemotherapy.     

A Korean female patient with thiamine-responsive pyruvate dehydrogenase complex deficiency due to a novel point mutation (Y161C)in the PDHA1 gene

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1alpha subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1 x 10(-3) mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.     

Measurement of serum chorionic gonadotropin by a biotin-avidin labeled enzyme immunoassay

A biotin-avidin enzyme immunoassay (EIA) for the measurement of human chorionic gonadotropin (hCG) in serum is described. This procedure involves the use of specific antibody immobilized on beads, biotin-labeled specific antibody, and enzyme-labeled avidin. Reproducible results were achieved within three hours for hCG in serum in the range of two mIU/ml to 150 mIU/ml. HCG levels as low as 0.4 mIU/ml can be measured. The biotin-avidin EIA and two commercially-available radioimmunoassay (RIA) kits were used to determine serum hCG levels on a group of patient samples. Good agreement was found between the biotin-avidin EIA and the RIA methods.     

Isolation and characterization of human monoclonal autoantibodies to glutamic acid decarboxylase

Production of human monoclonal autoantibodies to glutamic acid decarboxylase M(r) 65,000 (GAD65), characterization of their isotype, binding affinity, V region sequences and competition with autoantibodies in patients' sera is described. Lymphocytes from a patient with Addison's disease who had GAD65 autoantibodies without diabetes were immortalised and fused to a mouse/human hybridoma. In addition, mouse monoclonal antibodies to GAD65 were produced using standard techniques. F(ab')2S from our monoclonals and the GAD6 mouse monoclonal were used in competition with intact monoclonals and sera from diabetic patients for binding to 125I-labelled GAD65 (amino acids 46-586). Reactivities of the human monoclonals with GAD 65,000/67,000 M(r) chimeras were also studied. Variable region genes of human monoclonals were sequenced and analysed. The human monoclonals (n = 3) had affinity constants for GAD65 of 2.2 x 10(9), 5.8 x 10(9), 1.3 x 10(10) mol/l(-1); affinities of the mouse monoclonals (n = 5) ranged from 1.1 x 10(8) to 5.4 x 10(10) mol/l(-1). The binding of each of the human monoclonals was inhibited by GAD6 F(ab')2 and the binding of GAD6 antibody was inhibited by the human monoclonal F(ab')2S suggesting that the epitopes for these antibodies were overlapping. Studies with GAD65/GAD67 chimeras indicated that the human monoclonals reacted with C-terminal epitopes. The human monoclonals, GAD6 and 3/5 mouse monoclonals inhibited serum autoantibody binding to 125I-labelled GAD65. Overall, the human monoclonals were of high affinity, reacted with C-terminal epitopes and showed evidence of antigen driven maturation; they represented only a proportion of the repertoire of autoantibodies to GAD65 in the donor's serum and in the sera of patients with type-1 diabetes.     

Molecular heterogeneity of human chorionic gonadotropin in serum and urine from patients with trophoblastic tumors

Summary Many studies on the structure and function of human chorionic gonadotropin (hCG) have relied on purified hCG preparations obtained from pregnancy urine. In the present studies, in order to demonstrate possible differences between the hCG species present in serum and those released into urine, we examined serum and urinary samples derived from patients with trophoblastic tumors and of pregnancy origin by sodium dodecyl sulfate electrophoresis, isoelectric focusing, and two-dimensional electrophoresis including immunostaining with a specific hCG antibody and densitometry of the protein bands. This type of analysis was presently feasible only in patients presenting with extremely high serum levels of hCG and was therefore limited to seven patients with testicular cancers, one woman with a hydatidiform mole, and one pregnancy sample. We found marked differences in the isoelectric focusing pattern between urinary and serum hCG samples, with the urinary hCG consisting of more alkaline pl variants than that present in the serum of the same patients. Tumor hCG differed from pregnancy hCG in that it contained more acidic variants, and this was true for urinary and serum-derived materials. In some tumor and pregnancy samples an hCG immunoreactive material of lower molecular weight than hCG itself was found. In conclusion, the present studies, extending previous findings on the micro-heterogeneity of hCG, indicate that serum and urinary-derived hCG may differ in the composition of the isoform spectrum, as does tumor and pregancy hCG. Further, in some patients hCG immunoreactive molecules exist that differ markedly from hCG in size and charge. These observations suggest that, whenever possible, serum-derived hCG materials should be used to define the molecular structure of hCG and assess its biological activities.Abbreviations hCG human chorionic gonadotropin - mAb monoclonal antibody - SDS sodium dodecylsulfate Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

A typical postcesarean epithelioid trophoblastic lesion with placenta increta: Case report and literature review

We report an extremely rare case of unusual atypical epithelioid trophoblastic lesion from placenta increta. A 25-year-old Chinese woman with a history of 1 cesarean delivery was admitted to the hospital at 37 weeks’ gestation. Magnetic resonance imaging (MRI) showed the feature of placenta increta. An elective primary cesarean section was scheduled and a healthy baby was born. The patient was suggestive of placenta increta and implantation site was resected. Histology indicated a lesion consisting of epithelioid trophoblastic cells with an intermediate pattern between a classical placental site nodule and an epithelioid trophoblastic tumor. This may be linked to her previous cesarean delivery, which supports the relationship between atypical epithelioid trophoblastic lesion formation and surgical interventions. Her serum human chorionic gonadotropin (hCG) was 352.4 IU/L after delivery and gradually decreased to normal level.     

An unusual cause of elevated serum total beta hCG

Human chorionic gonadotropin (hCG), a heterodimeric hormone consisting of an alpha (alpha) and a beta (beta) subunit, is used as a marker for the diagnosis of pregnancy, congenital defects, and choriocarcinoma. After excluding the common causes of elevated serum hCG, laboratory identification of false-positive or true results assists in guiding clinical management. Options include testing urine for hCG, serum for heterophile antibodies, and serum hCG by different immunoassays. We report the case of a non-pregnant patient with chronic renal failure who had a positive urine hCG test, an elevated serum hCG level by two different assays but normal by a third assay, and persistently elevated serum hCG levels after ruling out the likelihood of heterophile antibodies. The discrepancies were explained by the patient's impaired renal clearance and the molecular forms of hCG that were measured by each assay. This case illustrates the importance of the laboratory's role in understanding the causes of elevated serum hCG.     

Differential associations of residual estradiol levels with bone mineral density and serum lipids in postmenopausal women with osteoporosis

OBJECTIVES: To examine the associations of residual endogenous estradiol (E2) to bone mineral density (BMD) and lipid concentrations in elderly women. METHODS: Subjects consisted of 59 elderly postmenopausal women with vertebral or femoral osteoporosis. BMD was measured at L2-4 and femoral neck by dual-energy X-ray absorptiometry (DEXA). Residual E2 concentrations were assessed by a sensitive radioimmunoassay. Data were expressed as mean +/- S.E.M. RESULTS: The age of the subjects was 65.2 +/- 0.8 years with 18.9 +/- 1.0 years postmenopausal. The mean residual E2 concentration was 6.0 +/- 0.5 pg/ml. There was a correlation between E2 levels and BMD at L2-4 (r = 0.32, P < 0.01) while no association was found at the femoral neck. The association between E2 and L2-4 BMD persisted after adjusting for years since menopause and body weight (r = 0.33, P < 0.05). With regard to serum lipid concentrations, no association of serum total cholesterol, LDL-cholesterol, HDL-cholesterol or triglyceride concentrations with residual E2 was found. CONCLUSIONS: Our findings confirm the role of residual endogenous E2 in the determination of bone mass in postmenopausal women with osteoporosis. The effect of residual E2 appears to be skeletal specific and possess no association with serum lipid concentrations.     

Epithelioid trophoblastic tumor: clinicopathological features with an emphasis on uterine cervical involvement.

We report on the clinical and histological features of five cases of epithelioid trophoblastic tumor, with an emphasis on its involvement of the uterine cervix. All five patients were of reproductive age (median age 38.4 years) and all, except one, presented with vaginal bleeding 3 to 18 years after the most recent pregnancy. One patient presented with amenorrhea. Elevation of serum human chorionic gonadotropin (hCG) was seen in four cases. Pathologically, the tumor involved endocervix in three cases and involved uterine corpus in another two. All five tumors were invasive, nodular lesions consisting of epithelioid intermediate trophoblastic cells that were mononuclear with abundant eosinophilic cytoplasm, along with zones of hyaline material and necrotic debris. In three cases of cervical involvement, the neoplastic cells focally replaced endocervical surface and glandular epithelium, simulating high-grade squamous intraepithelial lesions. Immunohistochemically, all five tumors displayed focal positivity for human placental lactogen and hCG. Positive nuclear staining of p63 was seen in all five cases. All patients received total hysterectomy and various regimes of adjuvant chemotherapy. Three patients survived the tumor with no recurrences or metastases with follow-up periods of 3, 7 and 16 years. One patient is currently alive with lung metastasis 1 month after the surgery. One patient died of tumor metastasis 8 months after the diagnosis. In summary, with its unusual ability to simulate an invasive squamous cell carcinoma and other epithelioid neoplasms, epithelioid trophoblastic tumor frequently poses a diagnostic challenge, especially when involving the uterine cervix. High index of suspicion and an awareness of elevation of serum chorionic gonadotropin are crucial in reaching a correct diagnosis.